Rheumatoid arthritis (RA) is the most common inflammatory joint disease of autoimmune etiology. Characteristic histologic features in RA joints are synovial hyperplasia, inflammatory cell infiltrates, bone erosions and fibrin deposits. Carboxypeptidase B (CPB) is well established to play an anti-fibrinolytic role by removing C-terminal lysine residues from fibrin, thereby preventing its cleavage by plasmin. Recently, C3a, C5a, thrombin-cleaved osteopontin (OPN-R) and bradykinin were identified as additional substrates of CPB. These substrates are involved in inflammatory pathways including chemotaxis and T cell activation. CPB removes arginine residues from the C-terminus of these substrates and is thereby postulated to modulate biologic function of these inflammatory mediators. We discovered that CPB deficient mice developed dramatically more severe arthritis as compared to wild type (WT) controls following transfer of anti-collagen antibodies. C5 deficient mice were also protected from arthritis, while bradykinin B2 receptor deficient mice developed arthritis equivalent in severity to that in WT mice. CPB deficient mice exhibited increased leukocyte chemotaxis in an acute peritonitis model, and in vitro chemotaxis assays showed that thrombin-cleaved OPN and C5a act as chemoattractants. Genetic association studies using tag single nuclear polymorphisms (SNPs) representing the CPB2 gene showed that the minor allele SNP rs1409433 was associated with a reduced RA disease susceptibility. The non- synonymous C1064T CPB SNP is in strong linkage disequilibrium with rs1409433, and the C1064T variant encoding CPB Thr325Ile has been reported to possess a longer half-life (Schneider et al, 2002). Our overriding hypothesis is that CPB plays a central role in down- regulating inflammatory responses in RA. We hypothesize that CPB cleaves C5a and OPN-R to inactivate their chemotactic properties, and thereby reduces inflammatory responses that contribute to the development and progression of RA. Finally, we hypothesize that individuals possessing the C1064T CPB variant express CPB Thr325Ile which possesses a longer half- life, and that long half-life CPB is associated with reduced incidence and severity of RA. This proposal is to investigate the role of CPB in RA: Aim 1 will utilize murine models of RA to further characterize the mechanisms by which CPB modulates inflammatory arthritis;Aim 2 will determine if CPB cleavage of C5a and OPN-R inactivates their chemotactic properties;Aim 3 will investigate the genetic influence of CPB in human RA;and Aim 4 will characterize CPB protein expression in human RA. PUBLIC HEALTH RELEVANCE: Rheumatoid arthritis (RA) is a chronic autoimmune arthritis that affects 0.6% of the world population. Current therapies are only partially effective, and there is great need for more effective therapeutic approaches. Success of the proposed experiments would demonstrate that CPB is a natural down-regulator of inflammation in RA. The proposed experiments will provide insights into pathogenesis, and could lead to development of new therapeutic approaches for RA.